The basis of the Wiesbaden Museum was formed by the collections of Johann Isaak Freiherr von Gerning, which, following a suggestion by Johann Wolfgang von Goethe, were acquired by the Duchy of Nassau and initially made accessible to the public in the Erbprinzenpalais. Three museums were created, whose sponsors were the Nassauische Verein für Altertumskunde und Geschichtsforschung, the Nassauische Verein für Naturkunde and the Nassauische Kunstverein e.V.. They became municipal property in 1900 and were presented in a new museum building designed by architect Theodor Fischer from 1920 onwards.

In the design of the Fischer building, half of the picture gallery was intended for temporary exhibitions. In the 1920s and early 1930s, the Nassauischer Kunstverein developed a lively exhibition activity there. Important additions to the collection in the field of classical modernism were also made with the help of Wiesbaden citizens. These included numerous paintings on permanent loan from the Wiesbaden collector Heinrich Kirchhoff.

After the Nazi seizure of power in 1933, all of the paintings from the Expressionist and Constructivist movements were initially taken into storage and then transferred to the central collection storage facility in Potsdam in 1937. Kirchhoff had to take back his permanent loans. Hermann Voss was director of the museum from 1935-45. Under his leadership, over 200 works were acquired, including top-class Baroque paintings. From 1943, Voss was also a special representative for the Führer Museum in Linz. The legal provenance of his acquisitions is currently being investigated.

After the end of the war, the museum became the Americans' Central Collecting Point Wiesbaden. After the art treasures temporarily stored here, including Nefertiti, were returned to their owners, a phase of refurnishing the Museum Wiesbaden began in the 1950s and 1960s with the most economical means. It was during this period that the then museum director Clemens Weiler began to build up what is now the museum's most important collection - the works of Alexej von Jawlensky. Over the past 25 years, it has been expanded in terms of quality and selection of works to become the world's most important collection of this artist. The Classical Modernism department was given additional weight by 30 paintings and drawings of the highest quality from the estate of Hanna Bekkers vom Rath, which the Verein zur Förderung der Bildenden Kunst in Wiesbaden acquired in 1987 and made available to the Museum Wiesbaden on permanent loan. Another focus of the museum is constructive art. The estate of the artist Friedrich Vordemberge-Gildewart (1899-1962), which was transferred in 1997, and the collection of works by the Russian Eduard Steinberg, which has been in the Museum Wiesbaden since 2013, are particularly noteworthy here. In 1962, the Museum Wiesbaden became the starting point of the Fluxus movement.

After the Museum Wiesbaden was transferred to the state of Hesse in 1973, the originally independent three museums became three museum departments in organizational terms. In connection with the plans to establish a city museum, the Nassau Antiquities Collection was returned to municipal ownership in spring 2010. Today's two museum departments, the Art and Natural History Collections, have over 7,000^m2 of exhibition space at their disposal.

In 2007, the German section of the International Association of Art Critics AICA (Association Internationale des Critiques d'Art) named the Museum Wiesbaden "Museum of the Year" for its exhibition activities and its work in the field of modern art. The museum owes this recognition to the sharpening of its collection profile with a focus on Jawlensky and his surroundings, Constructivism in the context of Friedrich Vordemberge-Gildewart and the so-called silent avant-garde since the 1960s.

Following the completion of a comprehensive refurbishment of the two wings of the building, the Old Masters in the south wing and the permanent exhibition of the Natural History Collections entitled "Aesthetics of Nature" in the north wing of the museum were reopened in May 2013.